Anaerobic fermentation and aerobic respiration have been the two metabolic modes of interest for the industrial production of chemicals. Oxygen rich respiration offers very efficient cell growth (growth rate and yield) and converts a high percentage of the carbon source into carbon dioxide and cell mass (see Table 1). Anaerobic fermentation, on the other hand, results in poor cell growth and the synthesis of several fermentation products at high yields (e.g. lactate, formate, ethanol, acetate, succinate, etc.).
TABLE 1RESPIRATORY VS FERMENTATIVE METABOLISMAnaerobicAnaerobicAerobicVariableFermentationRespirationRespirationGrowth RateLOWIntermediateHIGHCell MassLOWIntermediateHIGHProduct YieldsHIGHHigh/IntermediateLOWCapital CostLOWLOWHIGHEnergy InputLOWLOWHIGH
Producing chemicals via oxygen rich processes, however, is more costly than using anaerobic methods for two reasons. First, aerobic fermenters are more expensive to build, due to both the higher cost per unit and the need for smaller fermenters with reduced economy of scale. Secondly, the aerobic fermenters are more costly to operate than their anaerobic counterparts due to low solubility of oxygen, which in turn requires high energy input to ensure appropriate supply of oxygen to the cells. This is especially relevant for the production of commodity chemicals, where fermentation costs can represent 50-90% of the total production cost.
Therefore, anaerobic methods are usually preferred where possible, and it is typical to grow cells to a large number aerobically, and then switch the cells to anaerobic culture for the production of desired molecules. Often, however, the method is unsuccessful, resulting in poor yields and rates.
It has been known for a long time that a mixture of several acids, including succinic acid, is produced starting with the fermentation of E. coli, as described by J L Stokes in 1949 in the article entitled “Fermentation of glucose by suspensions of Escherichia coli” published in J. Bacteriol., 57: 147-158. However, for each mole of fermented glucose, only 0.3 to 0.4 Moles of succinic acid are produced by the Stokes method.
Thus, to improve the yield, bacteria have been genetically modified so as to inactivate the metabolic pathways that consume the NADH necessary for the production of succinic acid and to activate the metabolic pathways for producing succinate (a salt of succinic acid). In fact, the fermentative metabolic pathway that allows the conversion of oxaloacetate to malate, then fumarate, and finally to succinate requires two moles of NADH per mole of succinate produced. The major metabolic obstacle for the production of succinate is therefore the cellular bioavailability of NADH.
In order to solve this problem, U.S. Pat. No. 7,223,567 describes the use of a recombinant E. coli strain which overproduces succinate for the same quantity of available NADH. This E. coli strain “SBS550MG-pHL413” has inactivated adhE, ldhA genes (involved in the pathways which consume NADH), inactivated ack-pta genes and the iclR gene (which activate the glyoxylate pathway), and contains a plasmid vector overexpressing an exogenous pyc gene.
The article by Sanchez et al. (entitled “Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity” in Metabolic Engineering 7 (2005) 229-239), and U.S. Pat. No. 7,223,567 and US2005042736 have each developed novel culture and production conditions associated with this strain in order to improve its succinic acid production yields. These patents are incorporated by reference in their entirety herein.
In Sanchez et al., U.S. Pat. No. 7,223,567 and US2005042736, SBS550MG-pHL413 is first cultured under aerobic conditions in an Erlenmeyer flask in an LB culture medium in order to produce a maximum amount of biomass, the biomass is concentrated by centrifugation and then transferred under anaerobic conditions into a bioreactor with a rich culture medium to produce succinic acid. In these experiments molar yields of succinate per mole of glucose reached as high as 1.2 or 1.3 in US2005042736, but were generally less than 1.5, but reached as high as 1.7 in U.S. Pat. No. 7,223,567.
However, these experimental culture conditions were developed on a laboratory scale and may be difficult to transpose to an industrial scale, because the first culture step in an Erlenmeyer flask and the centrifugation for recovering the concentrated biomass is not easily adapted for the handling of large volumes. Further, it would be desirable to increase the succinic acid yield and rate of this strain even more, and to make the high succinate yields more reproducible in large scale.
What is needed in the art is a novel culture method that allows high yield and rates of production of compounds, such as succinate, yet is amenable to scale up and is cost effective.